Poster Title: Rational Methyltransferase Expression in Escherichia coli for Transformation of New Organisms

Abstract: “Many organisms naturally possess complex physiological traits that are of interest for biotechnology research. The ability to easily harness these traits in their native host could usher in a new era of biotechnology where synthetic biology is routinely applied to these non-standard organisms. However, many of these organisms are unable to be bioengineered due to a lack of available genetic tools. The development of genetic tools is limited in part by the inability to efficiently transform DNA into these organisms. One of the major barriers to successful transformation of bacteria is native DNA restriction-modification systems. DNA restriction-modification systems act as a bacterial immune system to cut DNA that is methylated differently than the host and are typically comprised of methylation and restriction subunits. To prevent host death, restriction enzymes and the cognate DNA methylases recognize the same target sequence. Therefore, in order to overcome restriction, DNA needs to be methylated in the same manner as the host organism prior to transformation. In order to determine the sites targeted for restriction in these strains, methylome analysis was initially performed for 17 organisms in collaboration with the Department of Energy Joint Genome Institute. This information was used to choose methyltransferases for expression in E. coli using a new system for multiple gene integration in E. coli. The gene integration system utilizes serine bacteriophage integrases, which enable a single, unidirectional recombination event between two specific DNA sequences, attB and attP, for stable insertion of DNA into the E. coli chromosome. This process of mimicking host methylation patterns successfully allowed for transformation of the type strain of Clostridium thermocellum, strain ATCC 27405. The native methyltransferase gene Cthe0519 and a bi-functional Phi3TI methytransferase were expressed from the E. coli chromosome. Plasmid DNA isolated from this methylating E. coli strain was then transformed into C. thermocellum. A similar approach is being used to demonstrate genetic transformation of other phylogenetically and metabolically diverse organisms. This system will allow for rapid transformation of new organisms to help further bioengineering research.”

“Atomic force microscopy (AFM) has been one of the premier surface characterization tools the past 20 years. The multiplicity of techniques available on the AFM, ranging from mechanical to electrical measurement modes, is important to characterizing physical properties of materials across many industries, including semiconductor and medical fields. In these fields, AFM is useful but provides no insight to the inherent chemical changes in a sample.1,2 Certain materials are sensitive to electrochemical processes, such spontaneous polarization behavior in ferroelectrics,3 and investigation of these phenomena through a combined AFM/mass spectrometer can provide insight. In this work, we utilized a combined time-of-flight secondary ion mass spectrometer (ToF-SIMS) and AFM to understand the chemical effects in the AFM tip-surface junction after scanning in contact mode. After scanning, silicon was observed local to the scanned region on the surface, and this work explores the source and control of this deposition.

Firstly, different manufacturer tips were used while scanning PbZr0.2Ti0.8O3 (PZT) and SrTiO3 (STO) films in contact mode, two coated silicon tips (NanoSensors PPP-EFM and ND-MDT DCP 01) and a completely metal platinum-iridium tip (RMNano 25PtIr300B). Each tip was used to scan a 10×10 μm2 area in contact mode. To eliminate influence of surface adsorption layer, an oxygen ion sputtering gun was used to clean the films in-situ. A ToF-SIMS spectra shows a silicon signal localized to the scanned region, even with a pure metal tip. New tips were then investigated in the ToF-SIMS where silicon contamination was observed on the surface.

Second, to understand the deposition of the silicon, scanning parameters (contact force, applied electrical bias, and scanning velocity) were varied while scanning. After scanning, the samples were then switched to the ToF-SIMS to observe changes of local surface chemistry. ToF-SIMS investigation of the surface revealed no changes in the underlying sample chemistry. Multivariate analysis of data shows layers of silicon deposition in scanned region, which is confirmed using a high spectral resolution ToF-SIMS imaging. Of the three parameters, scanning speed showed the only marked changes in deposition of the silicon on the surface. Inverting the velocity gives a linear relationship of relative silicon intensity as a function of dwell time.

In samples and tips without a silicon signal, a small amount of silicon was deposited locally onto the surface after scanning with an AFM. High resolution mass spectra in the ToF-SIMS were used to confirm the contaminant as silicon. An investigation of the source shows the silicon is from contamination of the surface of the tips, most likely from atmospheric siloxanes in the packaging and storage materials for AFM tips. Variation of scanning parameters show dwell time is the main contributing factor for deposition, likely being a diffusion process. Understanding the chemical effects of scanning with an AFM may influence analysis of future AFM data, as some processes are chemically sensitive and a few layers of silicon can cause additional effects or noise. Cleaning the tips with an ion beam may be used should the contamination interfere with scans.”

Paper Title: Digging Deeper – Development and evaluation of a nontargeted metabolomics approach to identify biogeochemical hotspots in Arctic soils

Abstract: “Arctic soils contain twice as much carbon as what exists in the atmosphere and are warming twice as a fast as any other landscape on the planet. Rising temperatures are increasing permafrost thaw, both in depth and duration, threatening to increase microbial decomposition of soil organic matter and the release of CO2 and CH4 to the atmosphere. Reliably modeling where and when this release will occur requires knowledge of the chemical composition of soil organic matter; in particular, the most vulnerable or bioavailable fraction—low molecular weight (LMW) dissolved organic matter (DOM). Our understanding is limited, however, by the wide-ranging physicochemical properties and high fluxes of these compounds, posing significant challenges in detection, isolation, and quantification. The objective of this work was to establish a robust workflow, from sample collection to analyte annotation, to characterize LMW DOM with soil depth and from beneath two vegetation types that dominate the Arctic landscape. To achieve this, we evaluated a multi-dimensional separation using both reversed-phase (RP) and hydrophilic interaction (HILIC) liquid chromatography (LC), coupled with nano-electrospray (nanoESI), high-resolution tandem mass spectrometry (HRMS/MS) in positive and negative ion modes. Features were clustered based on accurate mass and fragmentation pattern, resulting in a matrix of thousands of peaks per sample. Peak areas for features not observed in the blanks or controls were compared across all samples, statistically significant differences with depth or between vegetation cover were determined, and the resulting list of features was matched to online databases. Annotated classes of LMW DOM compounds included plant and microbial metabolites, organic acids, osmolytes, sugars, simple peptides, and heterocyclic compounds. Based on the chemical profile, we were able to distinguish between samples at each depth and between vegetation types, suggesting that a molecularly-resolved, data-driven approach could enable more reliable predictions of how biogeochemical processes occurring at the molecular-scale (e.g. plant-microbial competition for organic nutrients) impact carbon fluxes in the Arctic at the landscape-scale.”

“Though my current work focuses on environmental microbiology, I am actually a molecular geneticist by training. This combination has allowed me to take a deeper dive into the genetic basis that may determine the fate of toxic pollutants in our environment. Specifically, the pollutants addressed in this study are chlorinated solvents, which are commonly found in groundwater systems. While all of these chlorinated solvents are toxic, some are particularly noteworthy as they have been shown to be human carcinogens. Some of these toxins are naturally occurring, while most of them are derived through anthropogenic means. Fortunately, or perhaps fortuitously, a fascinating group of microorganisms has adapted to surviving in these chlorinated solvents, even to the point of breaking them down as the microorganisms grow and divide. However, these bacteria cannot clean up our environment by themselves, because they actually require unique cofactors to carry out this degradation process.

In this study, we identified a fragment of DNA that encodes an enzyme to make these required cofactors. Interestingly, this gene is actually harbored in a different bacterial species, meaning that multiple members of the microbial community must come together to clean up these toxic wastes spilling into our ecosystem. Remarkably, this project demonstrates energy derived from biological systems in multiple facets. Some microorganisms can degrade these toxic chlorinated solvents and in doing so, use them for energy. Meanwhile, other bacteria use different sources of biological energy to produce cofactors, a biologically-derived, value-added product similar to vitamins and antibiotics, to support this process of cleaning up our environment.”

Christine Ajinjeru

Paper title: The influence of rheology on melt processing conditions of carbon fiber reinforced polyetherimide for Big Area Additive Manufacturing

Abstract: “An approach was presented for determining the melt processing conditions of high temperature amorphous thermoplastics, specifically carbon fiber reinforced polyetherimide (PEI), for Big Area Additive Manufacturing (BAAM). PEI is a high performance thermoplastic that is attractive for various high temperature applications in the automotive and aircraft industry. For PEI to be processed successfully with BAAM, it must be stable over a range of temperatures and print conditions to ensure the final part possesses the desired strength and modulus. Under this approach, thermal properties are first analyzed to identify the lower and upper operating limits for the polymer and then extensive rheological characterization is carried out at selected temperatures within these bounds. This study investigates the effect of temperature, fiber loading, and processing environment on rheology in order to identify suitable process parameters for extruding carbon-fiber reinforced PEI on BAAM.”

Paper title: Development of a modeling framework to forecast power demands in developing regions: Proof of concept using Uganda

Abstract: “Accurate and detailed energy demand estimates are crucial to achieving adequate energy infrastructure planning. These estimates are often non-existent or deficient in many developing countries, and consequently, electricity supply is unreliable. A novel approach for estimating electricity demand is presented. Our approach uses a global geographical population database with 1km2 spatial resolution as the foundational input. The use of spatial population data is based on the premise that electricity consumption is dependent on where people are located. These population counts are converted to electrical customers to create spatial power demand data which can be mapped. The resulting power demand maps could be valuable for energy infrastructure planning. In this study, Uganda is used as a pilot case-study. Analysis suggests that an additional 1.5 GW of power generation capacity needs to be availed to meet the lowest power demand scenario. The methodology developed can be extended to other regions of interest.”

Christine’s poster can also be viewed on the fourth floor of Greve Hall.

Peter Shankles

Peter’s research poster can also be viewed on fourth floor of Greve Hall.

Abstract: “Additive manufacturing, or 3D printing, has been a cornerstone of the product development pipeline for decades, playing an essential role in the development of both functional and cosmetic prototypes. In recent years, the prospects for distributed and open source manufacturing have exploded, enabled by a growing library of materials, low-cost printers, and communities dedicated to platform development.

In this work, the production of multiple microfluidic architectures using a hybrid 3D printing-soft lithography approach is demonstrated and shown to enable rapid device fabrication with resolution high enough to take advantage of laminar flow characteristics. The fabrication process outlined here is underpinned by the implementation of custom design software that replaces computer aided design and slicer software. Devices are designed in the program by assembling parameterized microfluidic building blocks. The fabrication process and flow control within 3D printed devices were demonstrated with a gradient generator and two droplet generator designs, and 3D networks using bridge structures printed in a single motion rather than layers.”

“Agriculture has become a multifaceted industry as the production of biofuels and the need for both greater productivity and sustainability grows in importance. However, there is a finite amount of land available, and a portion of this land is unusable due to the presence of major pollutants, such as heavy metal contaminants. As the demand for agricultural land rises, the viability of crops within soils that were previously considered unusable has become more appealing. There is a possibility that with modifications to the rhizosphere, a plant species may be able to survive in conditions that would otherwise be toxic to the plant, increasing overall land availability for use in agriculture. This would allow the planting of biofuel crops within fields that may not be optimized for the growth of crops intended for consumption due to soil contamination, thereby avoiding the “food-for-fuel” tradeoff that has driven agronomic policy concerns in the corn-based ethanol industry. Wild strains of Cenococcum geophilum are being isolated and characterized for genetic analysis. These strains will then be screened with the heavy metals cadmium, lead, strontium, copper and zinc to determine susceptibility, and those strains demonstrating the greatest tolerance will be introduced to Populus in greenhouse experiments to determine the impact of fungal presence, if any, on the plant host. This research will aim to lead to a greater understanding of the exchange that occurs within a plant-fungal system and determine if there is an increase in the overall hardiness of the plant when the fungal species are present and how this varies with the genetic makeup of the symbionts. Looking to the future, this research will impact the direction of biofuel crop production as knowledge of the plant-fungal relationship increases, as well as potentially open new land for agricultural development.”

“Ionic liquid electrolytes are gaining widespread application as a gate dielectric used to control ion transport in functional materials. This letter systematically examines the important influence that device geometry in standard “side gate” 3-terminal geometries plays in device performance of a well-known oxygen ion conductor. We show that the most influential component of device design is the ratio between the area of the gate electrode and the active channel, while the spacing between these components and their individual shapes have a negligible contribution. These findings provide much needed guidance in device design intended for ionotronic gating with ionic liquids.”

Humaira Taz

This month’s featured research was presented by Humaira Tazat the Materials Research Society (MRS) Fall 2016 Meeting held in Boston, MA in November 2016. Her oral presentation in symposium EM2:Rare-Earths in Advanced Photonics and Spintronics was one of the top three graduate student presentations, for which they were awarded graduate student prizes sponsored by Thorlabs and Elsevier. The talk was titled “Novel Room Temperature Ferromagnetic Semiconductor: amorphous Fe-Dy-Tb-Oxide”. This research is also published in Scientific Reports (doi: 10.1038/srep27869).

Our collaborator at University of Tennessee-Chattanooga, Dr. Tatiana Allen, and I also presented a poster at the same meeting. The poster focused on the changes in the conductivity, cation states, and magnetic properties of the Fe-Tb-Dy-Oxide as a result of annealing cycles. This poster was a finalist for the best poster award.

“We report a class of amorphous thin film material comprising of transition (Fe) and Lanthanide metals (Dy and Tb) that show unique combination of functional properties. Films were deposited with different atomic weight ratio (R) of Fe to Lanthanide (Dy + Tb) using electron beam co-evaporation at room temperature. The films were found to be amorphous, with grazing incidence x-ray diffraction and x-ray photoelectron spectroscopy studies indicating that the films were largely oxidized with a majority of the metal being in higher oxidation states. Films with R = 0.6 were semiconducting with visible light transmission due to a direct optical band-gap (2.49 eV), had low resistivity and sheet resistance (7.15 × 10−4 Ω-cm and ~200 Ω/sq respectively), and showed room temperature ferromagnetism. A metal to semiconductor transition with composition (for R < 11.9) also correlated well with the absence of any metallic Fe0 oxidation state in the R = 0.6 case as well as a significantly higher fraction of oxidized Dy. The combination of amorphous microstructure and room temperature electronic and magnetic properties could lead to the use of the material in multiple applications, including as a transparent conductor, active material in thin film transistors for display devices, and in spin-dependent electronics.”

The ability to identify and characterize nuclear material in the wild is critical for nuclear security. While spectroscopic signatures have been cataloged for many of these materials, there has been less work done to understand how these signatures may be affected by environmental conditions.Marie’s current work examines uranyl fluoride, an important intermediate in the nuclear fuel cycle, using computational and spectroscopic methods to determine how the chemical and physical properties of the material change with environmentally-relevant perturbations in temperature and relative humidity.

“Uranyl fluoride (UO2F2UO2F2) is a hygroscopic powder with two main structural phases: an anhydrous crystal and a partially hydrated crystal of the same R3¯mR3¯m symmetry. The formally closed-shell electron structure of anhydrous UO2F2UO2F2 is amenable to density functional theory calculations. We use density functional perturbation theory (DFPT) to calculate the vibrational frequencies of the anhydrous crystal structure and employ complementary inelastic neutron scattering and temperature-dependent Raman scattering to validate those frequencies. As a model closed-shell actinide, we investigated the effect of LDA, GGA, and non-local vdW functionals as well as the spherically averaged Hubbard +U correction on vibrational frequencies, electronic structure, and geometry of anhydrous UO2F2UO2F2. A particular choice of Ueff=5.5Ueff=5.5 eV yields the correct U–Oyl bond distance and vibrational frequencies for the characteristic Eg and A1g modes that are within the resolution of experiment. Inelastic neutron scattering and Raman scattering suggest a degree of water coupling to the lattice vibrations in the more experimentally accessible partially hydrated UO2F2UO2F2 system, with the symmetric stretching vibration shifted approximately 47 cm−1 lower in energy compared to the anhydrous structure. Evidence of water interaction with the uranyl ion is present from a two-peak decomposition of the uranyl stretching vibration in the Raman spectra and anion–hydrogen stretching vibrations in the inelastic neutron scattering spectra. A first-order dehydration phase transition temperature is definitively identified to be 125 °C using temperature-dependent Raman scattering.”

“Genes express proteins in bursts of activity with periods of no activity between bursts. Burst dynamics are characterized by a burst size (duration of a burst) and burst frequency (number of bursts per time). During a burst the gene draws on a limited pool of reusable resource. Little is known about the relationship between burst dynamics and resource sharing. Here we made cell-sized reaction chambers (both PDMS plastic and POPC lipid vesicles) and observed bursting dynamics as the size of the resource pools was varied. When the size of the resource pool was increased, the number of protein made increased. This increase in protein was achieved by increasing the burst size not burst frequency. This may be due to the fact that the 100 different molecules needed to make protein became localized. Localized components suggest large transcriptional burst sizes are correlated with large translational burst sizes. This correlation is confirmed with in vivoE.coli data. Our results demonstrate the link between bursting dynamics and resource sharing.”

“Genes express proteins in bursts of activity with periods of no activity between bursts. Burst dynamics are characterized by a burst size (duration of a burst) and burst frequency (number of bursts per time). During a burst the gene draws on a limited pool of reusable resource. Little is known about the relationship between burst dynamics and resource sharing. Here we made cell-sized reaction chambers (both PDMS plastic and POPC lipid vesicles) and observed bursting dynamics as the size of the resource pools was varied. When the size of the resource pool was increased, the number of protein made increased. This increase in protein was achieved by increasing the burst size not burst frequency. This may be due to the fact that the 100 different molecules needed to make protein became localized. Localized components suggest large transcriptional burst sizes are correlated with large translational burst sizes. This correlation is confirmed with in vivoE.coli data. Our results demonstrate the link between bursting dynamics and resource sharing.”